Lentiviruses are complex retroviruses which, in addition to the common retroviral genes gag, pol and env, contain other genes with regulatory or structural function. The higher complexity enables the lentivirus to modulate the life cycle in the course of latent infection. A typical and well-characterized lentivirus is the human immunodeficiency virus (HIV), however, several animal lentiviruses have been described as well.
Viral vectors derived from lentiviruses are a useful tool for gene delivery. The ability of lentiviral vectors to deliver a gene into a broad range of rodent, primate and human somatic cells makes these vectors well suited for transferring genes to a cell for gene therapy purposes. Lentiviruses can infect terminally differentiated cells that rarely divide, such as neurons and macrophages, which renders them particularly useful for certain gene therapy applications requiring the transduction of non-dividing cells.
For producing recombinant lentiviral vectors packaging cell lines are used which supply in trans the proteins necessary for producing infectious virions. An important consideration in the construction of retroviral packaging cell lines is the production of high titer vector supernatants free of recombinant replication competent retrovirus (RCR). One approach to minimize the likelihood of generating RCR in packaging cells is to divide the packaging functions into at least two constructs, for example, one which expresses the gag and pol gene products and the other which expresses the env gene product. This approach minimizes the ability for co-packaging and subsequent transfer of the two genomes, as well as significantly decreasing the frequency of recombination between the viral genomes in the packaging cell to produce RCR. In the event recombinants arise, mutations or deletions can be configured within the undesired gene products to render any possible recombinants non-functional. In addition, deletion of the 3′ LTR on the packaging constructs further reduces the ability to form functional recombinants.
One of the major hurdles encountered in the art when producing a stable lentiviral-based packaging cell line is the inability to maintain high levels of expression of Gag/Pol proteins. This could be due to the inherent toxicity of some of the lentiviral proteins or to diminished protein expression from promoter silencing. Accordingly, packaging systems currently known in the art are either transient packaging systems or employ inducible promoters to minimize toxicity problems (Naldini et al., Science 272:263-267, 1996; Kafri et al., Journal of Virology 73:576-584, 1999). These approaches, however, are disadvantageous because they require considerable effort and time for lentiviral vector production. Furthermore, vector batches obtained from such systems will display a higher variability as compared to batches that would be obtainable from stable packaging cell lines. Furthermore, it is difficult to scale up lentiviral vector production from a transient system.